Enhanced-fluidity Liquid Chromatography Research Articles

A comprehensive exploration of diverse green analytical techniques and their influence in different analytical fields

AbstractThe primary goals of green analytical chemistry (GAC) involve the development of novel analytical technologies or the modification of existing methods to incorporate procedures with reduced reliance on hazardous chemicals. Several approaches can be employed to achieve these objectives, including the utilization of environmentally friendly solvents and reagents, shortening chromatographic separation times, and the miniaturization of analytical devices. The review summarizes the evolution of GAC with its specific principles and with special emphasis on recent applications in modern era. A detailed exploration of the choice of green solvents and their practical utilization in green liquid chromatography (LC) techniques for environmentally friendly analysis of pharmaceuticals has been discussed. An ordinary description with a tabular illustration of various modern green analytical techniques like micellar LC, superheated water chromatography, high‐speed LC, enhanced fluidity LC, two‐dimensional LC, and aspects of gas chromatography has been thoroughly provided for concepts with their mechanism and limitations. GAC provides potential applications for food analysis, environmental analysis of pharmaceutically active compounds in the aquatic environment with special emphasis on strategies for greening LC and impurity profiling, and so on. Numerous benefits, limitations, and challenges that are linked to the field of GAC are highlighted in this review. We pay special attention to new green approaches, such as automation, miniaturization, direct analysis techniques, and some initiatives like the National Environmental Methods Index Label for Analytical Eco‐Scale assessment. In spite of many promising advancements, the understanding is still limited and challenging.

Effect of dilution solvent and injection volume on the analysis of basic hydrophilic therapeutic polypeptide salts with pressurized carbon dioxide mobile phases

The chromatographic analysis of long-chain hydrophilic therapeutic peptides, with molecular weight mostly in the 3500–4500 Da range (31–34 amino acids), is explored with pressurized CO2 in the mobile phase. The optimal method was obtained on a Torus 2-PIC column, with a gradient elution of 50–90% co-solvent in CO2, which is relevant of enhanced-fluidity liquid chromatography (EFLC). Both UV (210 nm) and mass spectrometric detection modes were employed to assess the purity of the major peak and its resolution from impurities. Ten out of the eleven peptides in this set were basic, thus they were analyzed as acetate or trifluoroacetate salts. As significant peak distortion was observed in some cases, thorough examination of dilution solvent and injection volume was conducted to improve peak shape and resolution from impurities. Finally, the best injection volume was 1 µL, as any other volume (smaller or larger) yielded distorted peaks, and the best dilution solvent composition was the same as the mobile phase co-solvent (methanol comprising 5% water and 0.1 % methanesulfonic acid). However, not all peptide salts were fully soluble in this solvent so other alternatives (including more water in the dilution solvent), offering adequate dissolution but slightly inferior chromatographic performance should be chosen in such cases.

Comparison of enhanced fluidity liquid chromatography with liquid chromatography for enantioseparation of selected [formula omitted]-lactam derivatives

The present paper describes the study of eight racemic γ-lactam derivatives with the goal of comparing three modes of liquid chromatography which are the reversed-phase mode, the normal-phase mode and the polar organic solvent chromatography mode with super/subcritical fluid chromatography and enhanced fluidity liquid chromatography in extreme conditions i.e. when the CO2 percentage is practically zero. For this study two CPSs were chosen. Chiralcel OD-H was used in HPLC conditions and Chiralcel OD-H and Lux Cellulose-2 were used in SFC conditions. It is reported in this work that the introduction of chlorine substituent on polysaccharide-based selectors contributes to an improved enantioselectivity in supercritical fluid chromatography (SFC) compared to the electron-donating substituted phenyl carbamate column, allowing the complete separation of seven analytes out of the eight.The effect of enhanced fluidity liquid chromatography conditions, described as an intermediate mode between HPLC and SFC is also explored. In this case, the composition of the mobile phase starting at 80% CO2 was replaced with almost 100% ethanol at the end, this assay achieving further extension of the polarity range of the mobile phase in a single run and successfully enabling the simultaneous analysis and separation of the racemates. Under a mobile phase constituted of 99% ethanol, the resolution values of the γ-lactam derivatives on Lux Cellulose-2 CSP, were comprised between 1.91 and 3.44. High values of flowrates, ranging from 4 mL/min to 10 mL/min were also evaluated for some analytes on the highly retentive Lux Cellulose-2 CSP, leading to resolution equal to 6.24 for compound 6 and 4.64 for compound 8.

Analysis of flavonoids with unified chromatography-electrospray ionization mass spectrometry-method development and application to compounds of pharmaceutical and cosmetic interest.

In this project, we aimed at analysing flavonoid-type compounds with unified chromatography (joining supercritical fluid chromatography and enhanced fluidity liquid chromatography with carbon dioxide-methanol mobile phases covering a wide range of compositions) and diode-array and electrospray ionization mass spectrometric detection (UC-DAD-ESI-MS). First, the chromatographic method was developed for 9 standard flavonoid molecules from three different families (flavanols, flavanones and flavonols, glycosylated or not), with a strong focus on mobile phase composition to achieve the elution of a wide range of flavonoids with good chromatographic quality (efficiency and resolution). For this purpose, two stationary phases were selected (ACQUITY UPC2 DEA and Diol), and five different additives (formic acid, citric acid, phosphoric acid, methanesulfonic acid and ammonium hydroxide) were successively introduced in the methanol co-solvent. The composition containing 0.1% methanesulfonic acid in methanol was retained as it provided the best chromatographic quality together with the possibility of hyphenating the chromatography to mass spectrometry. The DEA column appeared to provide the best efficiency and was retained for further method development. The gradient method was then optimized to achieve a fast analysis, which involved elution with a wide range of mobile phase composition (from 20 to 100% co-solvent in methanol) together with reversed flow rate and reversed pressure gradients at fixed temperature. The final gradient lasted 10min, followed by 2.5min of re-equilibration. Then, ESI-MS detection was optimized. Because the single-quadrupole mass spectrometer employed (ACQUITY UPC2 QDa) allowed the variation of only a few parameters, a design of experiments was used to define the best compromise for three parameters (probe temperature, cone voltage and capillary voltage). The make-up fluid introduced before entering the MS was also varied: different compositions of methanol-water containing either formic acid, ammonium hydroxide or sodium chloride were tested. The best results in terms of signal-to-noise ratio were obtained with methanol containing 20mM ammonium hydroxide and 2% water. The optimal UC-DAD-ESI-MS method was then applied to two different flavonoid formulation ingredients. The first one, hidrosmin (5-O-(β-hydroxyethyl)diosmin), is known for its vasoprotective properties and therefore employed in pharmaceutical formulations. The second one, α-glucosyl-hesperidin (sometimes referred to as vitamin P), is employed in cosmetic formulations. Identification of the major compounds in each sample was achieved with the help of MS detection. Graphical abstract.

A chiral unified chromatography-mass spectrometry method to analyze free amino acids.

In this project, we aimed at analyzing native (or free) amino acids with supercritical fluid chromatography coupled to mass spectrometric detection, with modern instruments and methods, and maintaining as simple a mobile phase as possible to ensure applicability of the method. The purpose was twofold: (i) a generic method allowing for satisfactory elution of a wide range of amino acids (acidic, basic, or neutral residue) and (ii) resolution of the enantiomeric pairs. The Chiralpak ZWIX (+) and (-) stationary phases were selected as they are well-known for the enantioresolution of amino acids in liquid chromatographic modes. A wide range elution gradient, starting with a large concentration of carbon dioxide (90%) and finishing at 100% solvent (methanol containing 70mM ammonium formate and 7% water) allowed the elution of 18 native proteinogenic amino acids out of 19 injected. In these conditions, enantioselectivity was achieved for 16 of them. The basic amino acids (arginine, histidine, and lysine) were the most difficult to elute in these conditions, resulting in rather poor peak shapes. Cysteine was never observed in any of the conditions tested. Sample application was attempted with two food supplements, tablets containing a mixture of 17 proteinogenic amino acids and capsules containing taurine and theanine that were not present in the standards used for the method development. The sample preparation method was very simple, involving dissolution of the tablets and capsules in acidified water, filtration, and dilution with methanol. Mass spectrometric detection (electrospray ionization with single-quadrupole mass detection) allowed for unambiguous identification of most amino acids, except for the leucine and isoleucine isomers that were not separated by the generic gradient. The observation of taurine and theanine also suggests that the method should be generally applicable to other native amino acids than the proteinogenic amino acids selected for the development of this method. As peak shapes and signal-to-noise ratios could still be improved, further developments are wanted to upgrade this method. Due to the wide gradient (10 to 100% co-solvent in carbon dioxide), the method cannot truly be called either supercritical fluid chromatography (SFC) or enhanced-fluidity liquid chromatography (EFLC), but should be related to "unified chromatography" (UC), joining SFC and HPLC. Graphical abstract.

Enhanced fluidity liquid chromatography: A guide to scaling up from analytical to preparative separations

The evolution of supercritical fluid chromatography (SFC) instrumentation, improved detection capability, and expanded modifier range has led to extending the reach of SFC to the analysis of a broader spectrum of analytes beyond enantioselective separations. However, preparative SFC has yet to see the same technological revitalization, especially in regards to the purification of highly polar analytes. Enhanced fluidity liquid chromatography (EFLC) has been demonstrated as one of the ways to extend the applicable range of SFC instrumentation to highly polar analytes such as proteins, carbohydrates, and nucleotides. Despite recent applications of EFLC for challenging mixtures of hydrophilic metabolites and analogs, its viability in preparative purification, which is of great importance to the pharmaceutical industry, remains unknown. Herein, multiple chromatographic parameters that are critical to achieve feasible EFLC purification methods were investigated, including system pressure as a function of modifier composition (for several MeOH:H2O ratios), effect of diluent injection conditions on peak shape, and optimization of mass load with diluent composition. The usage of 50% acetonitrile or methanol diluents provided the most volumetric loading capacity. In the case of sucrose, leveraging higher analyte solubility in water proved to be more favorable than the volumetric loading capacity of diluents with higher organic content. In fact, an 80 mg injection of sucrose was possible on a 2 cm preparative HILIC column with minimal peak shape degradation. The combined information led to the successful demonstration of EFLC for the preparative separation of sugars using readily available MS-directed SFC instrumentation.

Separation and characterization of KRas proteins and tryptic peptides using enhanced-fluidity liquid chromatography tandem mass spectrometry.

Enhanced-fluidity, reversed-phase liquid chromatography was developed using custom instrumentation for separation and characterization of intact KRas proteins and tryptic peptides. The KRas, HRas and NRas function as GDP-GTP regulated binary switches in many signalling pathways, and mutations in Ras proteins are frequently found in human cancers and represent poor prognosis markers for patients. Mutations of the KRas isoform constitute some of the most common aberrations among all human cancers and intensive drug discovery efforts have been directed toward targeting the KRas protein. Separation and characterization of the KRas protein and tryptic peptides are helpful for exploring targeting, which has not been fully investigated using liquid chromatography-tandem mass spectrometry. EFLC-MS provided improved chromatographic performance compared to traditional HPLC-MS in terms of shorter analysis time, increased ion intensity and a shift to higher charge states for intact KRas proteins.

Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization.

Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO2 to conventional liquid mobile phases. The addition of liquefied CO2 increases diffusivity and decreases viscosity, which inherently leads to a more efficient separation. Herein, EFLC is applied to hydrophobic interaction chromatography (HIC) stationary phases for the first time to study the impact of liquefied CO2 to the chromatographic behavior of proteins. The effects of liquefied CO2 on chromatographic properties, charge state distributions (CSDs), and ionization efficiencies were evaluated. EFLC offered improved chromatographic performance compared to conventional liquid chromatography (LC) methods including a shorter analysis time, better peak shapes, and higher plate numbers. The addition of liquefied CO2 to the mobile phase provided an electrospray ionization (ESI)-friendly and "supercharging" reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in large-scale application.

Enhanced fluidity liquid chromatography of inulin fructans using ternary solvent strength and selectivity gradients

The value of exploring selectivity and solvent strength ternary gradients in enhanced fluidity liquid chromatography (EFLC) is demonstrated for the separation of inulin-type fructans from chicory. Commercial binary pump systems for supercritical fluid chromatography only allow for the implementation of ternary solvent strength gradients which can be restrictive for the separation of polar polymeric analytes. In this work, a custom system was designed to extend the capability of EFLC to allow tuning of selectivity or solvent strength in ternary gradients. Gradient profiles were evaluated using the Berridge function (RF1), normalized resolution product (NRP), and gradient peak capacity (Pc). Selectivity gradients provided the separation of more analytes over time. The RF1 function showed favor to selectivity gradients with comparable Pc to that of solvent strength gradients. NRP did not strongly correlate with Pc or RF1 score. EFLC with the hydrophilic interaction chromatography, HILIC, separation mode was successfully employed to separate up to 47 fructan analytes in less than 25 min using a selectivity gradient.

Protein separations using enhanced-fluidity liquid chromatography

Enhanced-fluidity liquid chromatography (EFLC) methods using methanol/H2O/CO2 and hydrophilic interaction liquid chromatography (HILIC) were explored for the separation of proteins and peptides. EFLC is a separation mode that uses a mobile phase made of conventional solvents combined with liquid carbon dioxide (CO2) in subcritical conditions. The addition of liquid CO2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis. TFA additive and elevated temperature were leveraged as key factors in the separation of a 13-analyte intact protein mixture in under 5min. Under these conditions EFLC showed modest improvement in terms of peak asymmetry and analysis time over the competing ACN/H2O separation. Protein analytes detected by electrospray ionization – quadrupole time of flight, were shown to be unaffected by the addition of CO2 in the mobile phase. Herein, the feasibility of separating hydrophilic proteins up to 80kDa (with transferrin) is demonstrated for CO2-containing mobile phases.

Gradient separation of oligosaccharides and suppressing anomeric mutarotation with enhanced-fluidity liquid hydrophilic interaction chromatography

Enhanced fluidity liquid chromatography using the hydrophilic interaction retention mechanism (EFLC-HILIC) is studied as an alternative separation mode for analyzing oligosaccharides and other sugars. These carbohydrates, which are important for the study of foods and biological systems, are difficult to comprehensively profile and either require a non-green, expensive solvent (i.e. acetonitrile) or derivatization of the analytes at the expense of time, sample loss, and loss of quantitative information. These difficulties arise from the diverse isomerism, mutarotation, and lack of a useable chromophore/fluorophore for spectroscopic detection. Enhanced fluidity liquid chromatography is an alternative separation method that involves the use of conventional polar solvents, such as methanol/water mixtures, as the primary mobile phase component and liquid carbon dioxide (CO2) as the modifier in subcritical conditions. The addition of liquid CO2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis and higher efficiency. This work illustrates an optimized EFLC-HILIC separation of a test mixture of oligosaccharides and simple sugars with a resolution greater than 1.3 and an analysis time decrease of over 35% compared to a conventional HPLC HILIC-mode analysis using acetonitrile/water mobile phases.

Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A “green” technique

A “green” hydrophilic interaction liquid chromatography (HILIC) technique for separating the components of mixtures with a broad range of polarities is illustrated using enhanced-fluidity liquid mobile phases. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquid CO2 to conventional liquid mobile phases. Decreased mobile phase viscosity and increased analyte diffusivity results when a liquefied gas is dissolved in common liquid mobile phases. The impact of CO2 addition to a methanol:water (MeOH:H2O) mobile phase was studied to optimize HILIC gradient conditions. For the first time a fast separation of 16 ribonucleic acid (RNA) nucleosides/nucleotides was achieved (16min) with greater than 1.3 resolution for all analyte pairs. By using a gradient, the analysis time was reduced by over 100% compared to similar separations conducted under isocratic conditions. The optimal separation using MeOH:H2O:CO2 mobile phases was compared to MeOH:H2O and acetonitrile:water (ACN:H2O) mobile phases. Based on chromatographic performance parameters (efficiency, resolution and speed of analysis) and an assessment of the environmental impact of the mobile phase mixtures, MeOH:H2O:CO2 mixtures are preferred over ACN:H2O or MeOH:H2O mobile phases for the separation of mixtures of RNA nucleosides and nucleotides.

Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms.

The potential of enhanced-fluidity liquid chromatography, a subcritical chromatography technique, in mixed-mode hydrophilic interaction/strong cation-exchange separations is explored, using amino acids as analytes. The enhanced-fluidity liquid mobile phases were prepared by adding liquefied CO2 to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO2 to methanol/water mixtures resulted in increased retention of the more polar amino acids. The "optimized" chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO2 mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO2 mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced-fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced-fluidity mobile phase separation was governed by a mixed-mode retention mechanism of hydrophilic interaction/strong cation-exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.

Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides

Hydrophilic interaction chromatography (HILIC) is a liquid chromatographic separation mechanism commonly used for polar biological molecules. The use of enhanced-fluidity liquid chromatography (EFLC) with mixtures of methanol/water/carbon dioxide is compared to acetonitrile/water mobile phases for the separation of nucleosides and nucleotides under HILIC conditions. Enhanced-fluidity liquid chromatography involves using common mobile phases with the addition of substantial proportions of a dissolved gas which provides greater mobile phase diffusivity and lower viscosity. The impact of varying several experimental parameters, including temperature, addition of base, salt, and CO 2 was studied to provide optimized HILIC separations. Each of these parameters plays a key role in the retention of the analytes, which demonstrates the complexity of the retention mechanism in HILIC. The tailing of phosphorylated compounds was overcome with the use of phosphate salts and the addition of a strong base; efficiency and peak asymmetry were compared with the addition of either triethylamine (TEA), 1,4-diazabicyclo [2.2.2] octane (DABCO) or 1,5-diazabicyclo [4.3.0] non-5-ene (DBN). DBN and DABCO both led to increased efficiency and lower peak asymmetry; DBN provided the best results. Sodium chloride and carbon dioxide were added to enhance the selectivity between the analytes, giving a successful isocratic separation of nucleosides and nucleotides within 8 min. The retention mechanism involved in EFL-HILIC was explored by varying the temperature and the mole fraction of CO 2. These studies showed that partitioning was the dominant mechanism. The thermodynamics study confirmed that the solvent strength is maintained in EFLC and that a change in entropy was mainly responsible for the improved selectivity. The selectivity using methanol/water/carbon dioxide varied greatly compared to that obtained with acetonitrile/water. Finally while this study highlights the optimization of EFL-HILIC for the separation of nucleosides and nucleotides under isocratic conditions, this is also an example of the broad range of polarities of compounds that EFL-HILIC can separate.

Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides

The application of enhanced fluidity liquid (EFL) mobile phases to improving isocratic chromatographic separation of nucleosides in hydrophilic interaction liquid chromatography (HILIC) mode is described. The EFL mobile phase was created by adding carbon dioxide to a methanol/buffer solution. Previous work has shown that EFL mobile phases typically increase the efficiency and the speed of the separation. Herein, an increase in resolution with the addition of carbon dioxide is also observed. This increase in resolution was achieved through increased selectivity and retention with minimal change in separation efficiency. The addition of CO 2 to the mobile phase effectively decreases its polarity, thereby promoting retention in HILIC. Conventional organic solvents of similar nonpolar nature cannot be used to achieve similar results because they are not miscible with methanol and water. The separation of nucleosides with methanol/aqueous buffer/CO 2 mobile phases was also compared to that using acetonitrile/buffer mobile phases. A marked decrease in the necessary separation time was noted for methanol/aqueous buffer/CO 2 mobile phases compared to acetonitrile/buffer mobile phases. There was also an unusual reversal in the elution order of uridine and adenosine when CO 2 was included in the mobile phase.

Separation of dimer acids using enhanced-fluidity liquid chromatography

The polymerization products of C18 fatty acids are complex mixtures of high dimeric, trimeric and higher molecular weight liquid acids. The current separation method for these mixtures involves a lengthy normal-phase HPLC gradient and the use of primary standards that are prepared via a semi-preparative HPLC. In this study, enhanced-fluidity liquid mobile phases, methanol/CO2 and methanol/CHF3, were studied as a possible alternative methodology for the separation of dimer and trimer acids. This study demonstrated that enhanced-fluidity liquid chromatography provides several advantages over normal-phase HPLC with conventional solvents. These advantages included faster speed of analysis, lower consumption of organic solvents, and much improved sample throughput.The best separation of dimer acids was achieved on a silica column using a steep methanol concentration gradient. Methanol/CO2 and methanol/CHF3 mixtures showed comparable elution strength using this steep gradient on the silica stationary phase. However, these polar compounds showed a normal-phase type retention on five reversed-phase columns which indicated an unusual retention mechanism.

Reversed-phase separation of basic tricyclic antidepressants using buffered and fluoroform-enhanced fluidity liquid mobile phases

In an effort to expand the range of applications of enhanced-fluidity liquid chromatography (EFLC) to strongly polar and basic analytes, fluoroform (CHF 3) was investigated as a fluidity-enhancing agent. Fluoroform was chosen due to its high polarity, low viscosity and chemical inertness toward water and basic analytes. A group of representative basic compounds, tricyclic antidepressants, covering a wide range of polarity was chosen as model compounds. Their retention behavior on a C 18 stationary phase in methanol/phosphate buffer and methanol/phosphate buffer/CHF 3 mobile phases was characterized. The chromatographic performance with mobile phase conditions of different pH, with and without CHF 3 addition and with addition of triethylamine was studied. The advantages of using CHF 3 enhanced and buffered mobile phases were shown in the much improved chromatographic performance, such as shortened analysis time, increased efficiency, lower pressure drop and improved selectivity. Furthermore, this study demonstrated for the first time, that a commercial instrument could be readily utilized for EFLC separations which greatly expands the application range of the EFLC technique and chromatographic instrumentation.

Chiral Separations Performed by Enhanced-Fluidity Liquid Chromatography on a Macrocyclic Antibiotic Chiral Stationary Phase

The use of enhanced-fluidity liquid chromatography (EFLC) for chiral separations was demonstrated on a macrocyclic antibiotic column, Chirobiotic-V. This technique was compared to high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) for the separation of chiral compounds in normal-phase mode. The highest resolution was always observed for EFLC condition. Higher efficiency and shorter retention time were also observed for most separations with portions of CO(2) in the range of 0-50 mol %. Larger amounts of CO(2) caused efficiency to decrease and retention time to be prolonged. For some separations, the temperature was elevated to bring the mobile phase to the supercritical condition. Improved efficiency was obtained in SFC, whereas resolution and selectivity were worse. The use of EFLC in reversed-phase chiral separations was also tested. Enantiomer resolution improved under the EFLC condition. For the tested methanol/H(2)O mixture, fluoroform provided more significant improvements in chromatographic performance than CO(2) when used as a fluidity enhancing liquid. The use of EFLC instead of HPLC also caused a markedly lower pressure drop across the column for commonly used flow rates. The low-pressure drop will allow the use of longer columns or multiple columns to increase the total efficiency of the separation. Since chiral columns are often inefficient, this attribute may be very important for chiral separations.

Phase diagram studies of methanol–CHF 3 and methanol–H 2O–CHF 3 mixtures

Comprehensive phase diagrams of methanol/CHF 3 and methanol/H 2O/CHF 3 mixtures over the temperature range of 25–100°C and pressure range of atmospheric to 340 atm are reported. Fluoroform is expected to be useful as an alternative to CO 2 for enhancing the fluidity of liquid mixtures due to its high polarity and low viscosity. Therefore, these mixtures will be studied as mobile phases for enhanced-fluidity liquid chromatography and extraction. The phase behavior of methanol/CHF 3 and methanol/H 2O/CHF 3 was compared to that of methanol/CO 2 and methanol/H 2O/CHF 3. Fluoroform is markedly more miscible with methanol or methanol/H 2O than is CO 2. Data for methanol/CHF 3 binary mixtures were also modeled by the Peng–Robinson equation of state. The correlation results showed that the PR equation of state with two temperature-independent binary parameters was capable of representing the experimental data over the entire temperature range with an average relative deviation within 6%.